A major obstacle to developing an efficacious TB vaccine is illustrated by the results of a recent clinical trial in which the vaccine induced polyfunctional T cell responses, yet it did not prevent TB. This obstacle is also illustrated in animal models, as existing TB vaccines induce T cell responses, but have only modest effects on bacterial burdens. These results suggest that antigen-specific T cells are generated by infection or vaccination and they can be activated by ex vivo restimulation, but they are not activated effectively at the site of infection. The long term objective of this project is to guide development of efficacious TB vaccines by: 1) characterizing a major mechanism that limits the ability of antigen-specific CD4 T cells to recognize M. tuberculosis-infected cells and become activated at the site of infection; and 2) developing solutions that overcome or bypass that mechanism. We have discovered that M. tuberculosis-infected cells activate CD4 T cells poorly because multiple secreted bacterial antigens are shunted away from the MHC class II antigen processing and presentation pathway and are exported from the infected cells. This novel mechanism, which we term antigen export, involves intracellular vesicular transport, and requires the microtubule-directed molecular motor, kinesin-2. When we deplete M. tuberculosis-infected cells of kinesin 2 and thus block antigen export, we find increased MHC class II antigen presentation and CD4 T cell activation by infected cells, resulting in improved control of intracellular M. tuberculosis. This indicates that CD4 T cells have the potential to exert effective antimycobacterial activity, but their activation is limited by poor antigen presentation by infected cells, and poor antigen presentation is secondary to antigen export. This project will extend these findings and characterize the other cellular mechanisms required for antigen export, including budding of antigen export vesicles (AEV) from phagosomes, intracellular targeting of AEV, and fusion of AEV membranes with the plasma membrane for release of antigens to the extracellular space. Our goal is to identify host molecules that can be targeted with drugs to block antigen export and make antigen-specific CD4 T cells more effective in TB. To inform TB vaccine design, we hypothesize that nonsecreted (and nonexported) antigens are more desirable than secreted antigens in TB vaccines, since secreted antigens are exported from infected cells, making the infected cells poor targets for recognition by CD4 T cells specific for those antigens. To test the hypothesis that CD4 T cells directed against a nonsecreted antigen are more efficacious than those directed against a secreted antigen, we have modified an immunodominant secreted M. tuberculosis antigen (Ag85B) so that it is not secreted, and we will determine whether bacteria that express nonsecreted-nonexported Ag85B are better controlled by CD4 T cells in vivo. Together, our proposed studies will guide efforts to make naturally-occurring and vaccine-induced CD4 T cells more effective in TB, and contribute to solving the problem of global tuberculosis.